Search Results (111)

The prevalence of diabetes has increased considerably in recent decades, representing a global health problem. Although several pharmacological approaches for the treatment of diabetes are available, it is pertinent to explore more effective alternatives with reduced adverse effects. In this work, the hypoglycemic activity of rhamnolipids was studied using the chicken embryo (in ovo) model. The results obtained demonstrated that rhamnolipids produced by Pseudomonas aeruginosa #112 and commercial rhamnolipids (RL-90) significantly reduced blood glucose levels of chicken embryos on embryonic day 11 (from 135 ± 11 mg/dL to 94–107 mg/dL). These values were similar to those achieved with human insulin (104 ± 20 mg/dL) and the rapid-acting human insulin analog FIASP^® (95 ± 18 mg/dL). It was also verified that rhamnolipids did not have a negative effect on chicken embryo development at the concentrations tested. Regarding the molecular mechanisms involved in the decrease in blood glucose levels, for both insulins, a reduction in the expression of genes encoding the glucose transporter 2 (glut2) and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase 2 and fructose-1,6-biphosphatase 1 was observed. However, in the case of rhamnolipids, only a reduction in the expression of glut2 was observed. According to the results obtained, rhamnolipids are potential candidates for further studies on the development of new alternative treatments for diabetes symptoms. Full article

Early microbial exposure during embryogenesis may shape post-hatch gut development in poultry, yet the effects of sublethal pathogenic exposure and in ovo synbiotics remain unclear. This model-establishment study preliminarily established in ovo Salmonella Enteritidis (SE) infection and synbiotic (SYN) delivery models and evaluated their effects on hatchability, cecal microbiota, intestinal morphology, epithelial turnover, and barrier function in newly hatched chicks. In one group, the air cells of specific pathogen-free White Leghorn eggs were injected with SE on embryonic day 12; in another group, a synbiotic consisting of Lactobacillus plantarum, Pediococcus acidilactici, and inulin was injected into the amniotic cavity on embryonic day 17.5. Sterile saline was injected as the vehicle-only procedural control at the corresponding time points and injection sites. Based on their impacts on hatchability, SE1-L and SYN-H were selected. SE1-L reduced cecal microbial diversity, expanded Proteobacteria and Escherichia–Shigella, increased ileal apoptosis and crypt depth, decreased the villus height-to-crypt depth ratio, downregulated jejunal tight-junction genes, upregulated ileal MYD88 and TNF-α, and increased plasma lipopolysaccharide and D-lactate. In contrast, SYN-H maintained hatchability, promoted early Pediococcus colonization, suppressed potential pathogens, increased ileal villus height and villus height-to-crypt depth ratio, enhanced proliferation, reduced apoptosis, and improved mucosal barrier-related indices. These findings provide preliminary evidence that embryonic SE infection and synbiotic delivery differentially influence early intestinal microbiota succession and gut development in newly hatched chicks. Full article

Lung cancer remains a leading cause of cancer-related mortality and is frequently complicated by respiratory infections, supporting interest in agents with both antitumoral and antimicrobial potential. This study evaluated two standardized thyme-derived, carvacrol-based formulations, Vacrol and S-Mix, in lung cancer-associated experimental models. A549 lung adenocarcinoma and BEAS-2B bronchial epithelial cells were treated with the formulations, and cell viability, clonogenic capacity, SIRT1–SIRT7 protein expression, in ovo tumor growth, histopathological changes, and antimicrobial activity against pneumonia-associated reference strains were assessed. S-Mix showed stronger short-term cytotoxicity in A549 cells, reaching an IC₅₀ of 1 mM after 72 h, whereas Vacrol produced more pronounced modulation of selected sirtuin proteins, particularly SIRT1, SIRT4, and SIRT5. Both formulations suppressed colony formation under prolonged exposure. In the CAM model, Vacrol was associated with greater macroscopic suppression of tumor growth and vascularization, while S-Mix produced more prominent histopathological cellular injury. Vacrol also showed antimicrobial activity against tested respiratory pathogens, with MIC values ranging from 0.5 to 4 mg/mL, MBC values ranging from 1 to 4 mg/mL and volatile-phase activity against Streptococcus pneumoniae as well as Klebsiella pneumoniae. These findings suggest that carvacrol-based formulations exert distinct cytotoxic, sirtuin-modulatory, antitumoral, and antimicrobial effects, warranting further mechanistic and translational validation. Full article

Preclinical multimodal imaging is widely applied in small animal models for longitudinal studies of human diseases. Beyond murine systems, cost-effective and ethically sustainable models such as the chicken embryo and its chorioallantoic membrane are gaining increasing interest in accordance with the 3Rs principles. This study evaluated the feasibility of using both high-frequency ultrasound and magnetic resonance imaging for the non-invasive longitudinal monitoring of chicken embryo development in ovo. Fifty fertilized eggs were incubated under controlled conditions and examined up to embryonic day 14. High-frequency ultrasound (15–71 MHz) enabled real-time imaging and quantitative assessment of superficial structures, including cranial biometry and limb growth, while magnetic resonance imaging (7T) provided high-resolution three-dimensional visualization of internal organs and extraembryonic compartments. Together, these modalities allowed the progressive identification of key anatomical structures from ED5 onward, with HFUS enabling earlier linear measurements and MRI facilitating detailed anatomical and volumetric evaluation. The integration of these techniques allowed the generation of a developmental imaging timeline and quantitative reference dataset of normal embryogenesis. This multimodal approach represents a promising strategy for in vivo developmental studies, offering a robust baseline to characterize structural alterations induced by experimental conditions. Moreover, the use of the chicken embryo model provides significant ethical and economic advantages, supporting its application in preclinical research and imaging-based studies. Full article

(1) Background: Diclofenac sodium is a drug with reported developmental toxicity in several non-mammalian and mammalian models. This study aims to evaluate the stage-dependent embryolethality of diclofenac sodium according to developmental stage using the chicken (Gallus gallus domesticus) embryo in ovo model, a system widely used in veterinary and avian developmental toxicology screening. The study focuses specifically on quantitatively determining the “critical sensitivity windows” between the early (day 7) and late (day 14) embryonic stages. (2) Methods: Fertilized chicken eggs (Gallus gallus domesticus) were exposed to different doses (3.125–50 mg/kg) of diclofenac sodium, and mortality rates were analyzed. (3) Results: The data indicated that diclofenac toxicity is highly dependent on the developmental stage (p = 0.002). While the LD50 value for the early stage (day 7) was calculated as 20.67 mg/kg, (95% CI 6.79–860.87 mg/kg; wide interval reflecting low precision and steep response), embryos at the late stage (day 14) were found to be more resistant, with an LD50 value of 32.16 mg/kg (95% CI 27.77–37.90 mg/kg). The calculated Potency Ratio of 1.55 indicates that 7-day-old embryos are more sensitive to diclofenac. (4) Conclusions: This study provides model-specific preliminary quantitative data on the stage-dependent embryo mortality profile of diclofenac sodium in chicken embryos. The higher mortality observed on day 7 is consistent with a window of susceptibility during early organogenesis that may be associated with immature detoxification pathways (e.g., hepatic CYP450) and active organic anion transport (e.g., Oatp1d1) reported in other developmental models. However, these mechanisms were not directly measured here. These findings contribute to comparative developmental toxicology and the One Health-focused assessment of NSAID exposure in animal species, emphasizing the importance of considering “timing of exposure” in developmental toxicity assessments. Furthermore, no extrapolation to mammalian pregnancy or human clinical guidelines is implied. Full article

Chronic obstructive and inflammatory lung diseases share overlapping clinical manifestations and spirometric features, complicating differential diagnosis and monitoring. In this study, we performed an integrative real-time proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS) breathomics analysis to assess whether exhaled volatile organic compound (VOC) profiles enable multiclass discrimination among bronchial asthma (BA), chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and lymphangioleiomyomatosis (LAM), with healthy individuals as controls. Breath VOC data from 843 subjects were analyzed using a stratified 70/30 train/test split. An ensemble feature selection strategy based on gradient boosting (XGBoost with SMOTE within cross-validation) identified stable VOC panels (top 25% selection probability), yielding 29 VOCs and 31 features including clinical covariates. On the independent test set, the VOC-only model achieved a macro-averaged one-vs-one (OvO) AUC of 0.866 (95% CI 0.829–0.903), while the combined model improved to 0.888 (95% CI 0.853–0.919), indicating modest value of clinical variables. Pairwise analysis demonstrated highest discrimination for CF (AUC up to 0.988), whereas BA and LAM showed lower sensitivity (<0.60), likely reflecting heterogeneity and limited sample size. Given differences in age, sex, BMI, and smoking status across cohorts, confounding effects were assessed, confirming that VOC signatures retain independent diagnostic information. Disease-specific VOC interaction networks revealed distinct remodeling patterns, with central metabolites not captured by univariate analysis. Overall, PTR-TOF-MS breathomics demonstrates proof-of-concept multiclass discrimination across chronic lung diseases. Full article

Graphene oxide (GO) is a promising nanocarrier for the delivery of oligonucleotides. It offers a high loading capacity, efficient cellular uptake, and surface functionalization. MicroRNA-21 (miR-21) is a well-characterized oncomiR commonly overexpressed in hepatocellular carcinoma (HCC). In HCC, miR-21 contributes to tumor progression, inflammation, and angiogenesis. In a previous in vitro study, we showed that GO alone induces the upregulation of pro-inflammatory and tumor-related genes in HepG2 cells. However, conjugation with an antisense miR-21 (GO-antisense miRNA 21) reverses this effect, suggesting a potential therapeutic application. This study aims to evaluate the antitumor and anti-angiogenic efficacy of the GO-antisense miR-21 nanosystem in ovo using the chick embryo chorioallantoic membrane (CAM) model. Fertilized chicken eggs (n = 4 per group) were randomized into untreated, GO-treated, and GO–antisense miR-21-treated cohorts. A dose of 200 μL (GO 10.0 µg/mL: antisense miR-21 5.0 pmol/mL) was administered intratumorally. Tumor size, volume, and vascularization were monitored through stereomicroscopy and histological analysis. The expression of inflammatory and tumor-associated genes (IL-8, MCP-1, TIMP-2, ICAM-1 and NF-kB) was assessed by quantitative PCR. Given its prominent response, IL-8 protein expression was further analyzed via immunofluorescence. To evaluate tumor-specific delivery, FITC-labeled GO was tracked by confocal microscopy. Our data revealed that treatment with unfunctionalized graphene oxide (GO) unexpectedly promoted tumor vascularization and led to a significant increase in tumor weight. This was accompanied by upregulation of inflammatory markers. In contrast, GO-antisense miR-21 significantly reduced the tumor volume and vessel density. It also successfully downregulated all target genes. Confocal imaging demonstrated preferential accumulation of the nanosystem within the tumor mass. Our results highlight the dual anti-inflammatory and anti-angiogenic effects of GO-antisense miRNA 21 in ovo and support its potential as a targeted nanoplatform for HCC treatment. Full article

The rapid growth of Digital Financial Services (DFSs), including what is occurring in Indonesia, necessitates evaluation methods that are capable of objectively and systematically handling multiple assessment criteria. Therefore, this study aimed to propose a hybrid FSS–CRITIC–TOPSIS framework for selecting optimal DFSs. Fuzzy soft sets (FSSs) were used to model uncertainty and subjectivity in criterion assessments. The Criteria Importance Through Inter-criteria Correlation (CRITIC) method determined the weights objectively based on the degree of contrast and inter-criteria correlation. Subsequently, the Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) method was used to rank the alternatives based on the closeness to the ideal solution. The incorporation led to a formally defined decision operator, $\mathcal{T}$, which mapped FSS to complete preference orderings while ensuring provable stability and strong discriminative properties. The framework was applied to five major Indonesian digital wallets, namely ShopeePay, GoPay, OVO, LinkAja, and DANA, as well as being evaluated across five criteria. This framework identified DANA as the optimal alternative, with a score of 0.9282, followed by ShopeePay (0.8354) and GoPay (0.6958). Comparative analysis with other methods showed a near-perfect ranking correlation ($\rho =0.9–1$), with a more proportional score distribution and ranking results that reflected actual conditions. Sensitivity analysis also confirmed robustness, with ranking changes remaining logically consistent underweight variations. In conclusion, the FSS-CRITIC-TOPSIS framework provided an effective, mathematically rigorous method for multi-criteria decision-making (MCDM) under uncertainty, which applied to digital wallet selection as well as potential extension to broader evaluation contexts supporting SDGs 8, 9, and 10. Full article

Preclinical imaging has recently been expanded through the use of ostrich embryos as an alternative in vivo model. In ovo experiments represent a promising substitute for conventional rodent-based animal testing. For artifact-free dynamic nuclear medicine imaging, reliable immobilization of embryos is essential. Although previous studies have demonstrated the feasibility of isoflurane anesthesia, the kinetics and uptake mechanisms of isoflurane in ostrich embryos remain insufficiently characterized. The aim of this study was to characterize gas exchange dynamics in ostrich eggs and to quantify isoflurane uptake using two complementary approaches: indirect consumption measurements in a closed system and direct quantification by serial blood sampling. Fourteen ostrich eggs, including seven fertilized and seven unfertilized specimens, were analyzed at developmental stages up to day 37 of incubation. Gas exchange was assessed in a sealed container using a clinical anesthesia gas monitoring system to measure oxygen consumption and carbon dioxide excretion. Isoflurane uptake was evaluated during exposure to concentrations of 2%, 4%, or 6%. In a separate experimental series, serial blood samples were collected during and after exposure to the same concentrations to determine systemic uptake. Fertilized embryos showed progressive increases in metabolic activity, with a maximal oxygen consumption and carbon dioxide excretion of 116 mL/h/kg and 93 mL/h/kg on day 37. Indirect measurements demonstrated isoflurane uptake rates of up to 1.1 mL/min at 6%, with proportionally lower values at 4% and 2%. Blood analyses confirmed systemic absorption, peak concentrations of 160 µg/mL, and detectable residual levels for up to 120 min after exposure. These findings refine in ovo imaging. Full article

Despite advances in periodontal regenerative therapies, consistent tissue regeneration remains challenging, with cells playing an essential role in successful repair. Therefore, this study tested different dental bone substitutes embedded in the chorioallantoic membrane (CAM) combined with periosteum-derived micrografts obtained using a chair-side device (Rigenera HBW system). Cell populations within the micrografts were identified and characterised via immunofluorescence and flow cytometry (CD31, CD105, CD34, CD90, CD73, and CD45). A CAM model was employed to examine the angiogenic potential of micrografts combined with bone substitutes, which were analysed through quantitative blood vessel/vascularisation assessments using the Ikosa software (2025), along with histological and immunohistochemical evaluations such as smooth muscle actin (SMA), H&E, and Masson’s trichrome staining. Statistical analysis was performed using GraphPad Prism 10. The addition of periosteum-derived micrografts resulted in angiogenic enhancement compared to the controls. Notable enhancement of total vessel area, total length, and branching points were obtained when Fisiograft^® (p = 0.0007, p = 0.0002, and p < 0.0001, respectively), New Shore^® (p = 0.0006, p = 0.0149, and p = 0.0083, respectively), and Bio-Oss^® (p = 0.0038 and p = 0.0010, respectively) were combined with micrografts, compared to the positive controls. The histological and immunohistochemical analyses confirmed increased vascularisation (positive staining for SMA) in the micrograft groups. Periosteum-derived micrografts represent a promising adjunct to conventional bone-grafting materials, promoting vascularisation and potentially enhancing tissue regeneration and healing outcomes. Full article

Background/Objectives: Dextran sulfate sodium (DSS) is widely used to induce intestinal injury, reducing intestinal barrier integrity and thus contributing to systemic inflammation and oxidative stress, which may affect liver homeostasis and central nervous system function. In this context, the intake of phenolic compounds and anthocyanins from fruits such as jabuticaba has gained attention due to their antioxidant and anti-inflammatory properties. This study evaluated the effects of jabuticaba in the form of freeze-dried whole fruit, freeze-dried peel, and microencapsulated peel extract on DSS-induced damage to the gut–liver–brain axis in an in ovo model. Methods: Fertile eggs were assigned to five groups: water, DSS, DSS plus whole jabuticaba (WJ), DSS plus jabuticaba peel (JP), and DSS plus microencapsulated jabuticaba peel (JM). Duodenal, colon, and liver gene expressions; and histomorphometry, cecal microbiota, and brain gene expressions were evaluated at hatch. Results: DSS administration increased NF-κB expression and reduced MUC-2 in the duodenum, induced colonic inflammation, altered cecal microbiota, and caused hepatic oxidative stress, evidenced by elevated iNOS and enlarged fat globules, while reducing brain BDNF levels. Jabuticaba treatments mitigated intestinal, hepatic, and neural damage by reducing inflammatory markers; enhancing MUC-2, ZO-2, JAM-2, and claudin-1 expression; increasing villus area and goblet cell numbers; normalizing CAT and SOD activities in the liver; decreasing COX-2; increasing dopamine; and restoring BDNF in the brain. Conclusions: This study demonstrates that jabuticaba exerts protective effects along the gut–liver–brain axis, highlighting its potential as a functional food to support intestinal, hepatic, and brain health. Full article

Naphthyridine derivatives have emerged as privileged scaffolds with diverse pharmacological activities, particularly in anticancer and antiviral drug discovery. In this study, a series of naphthyridine-based derivatives (1–10b) was designed, synthesized, and structurally characterized using IR, ¹H/¹³C NMR, and mass spectrometry, and evaluated as dual-function antiproliferative and anti-fowlpox virus agents supported by integrated computational analyses. The synthesized compounds were screened for in vitro antiproliferative activity against HeLa, HCT-116, and MCF-7 cancer cell lines, as well as normal WI-38 lung fibroblasts. Several derivatives exhibited potent cytotoxic activity with enhanced selectivity toward cancer cells. Compound 5b showed the highest activity against HeLa cells, compound 1 was most effective against HCT-116 cells, while compounds 7 and 8 displayed remarkable activity against MCF-7 cells, with compound 7 surpassing doxorubicin and compound 8 demonstrating excellent selectivity toward normal cells. Mechanistic investigations revealed that compounds 7 and 8 acted as dual topoisomerase I/IIβ inhibitors, inducing G2/M cell cycle arrest and intrinsic apoptosis associated with caspase-9 activation and downregulation of topoisomerase II protein expression. Selected derivatives were further evaluated for antiviral activity against fowlpox virus using in ovo and in vivo SPF embryonated chicken egg models, where compounds 2 and 9a exhibited the highest therapeutic indices, comparable to ribavirin, and compound 9a markedly suppressed viral replication and titers in vivo. ADMET profiling, molecular docking, molecular dynamics simulations, and DFT calculations supported the experimental findings and identified compound 10a as the most favorable theoretical candidate. Overall, this integrated experimental–computational approach establishes naphthyridine derivatives as a rationally designed multifunctional chemotype for simultaneous anticancer and antiviral drug development. Full article

Background/Objectives: Bladder cancer remains a therapeutically challenging malignancy due to high recurrence rates, progression to muscle-invasive disease, and frequent resistance to cisplatin-based chemotherapy. Cold physical plasma (hereafter referred to as plasma) has emerged as a locally applicable modality that generates reactive oxygen species (ROS) and shows preclinical antitumor activity, offering a potential strategy to enhance cisplatin efficacy while enabling dose reduction. Here, we investigated combination treatment with cisplatin and argon plasma generated by the clinically approved kINPen jet in human bladder cancer models. Methods: Three bladder cancer cell lines representing distinct entities were used, namely the urothelial carcinoma lines RT-112 and T24, and the squamous cell carcinoma line SCaBER. IC₂₅ values for plasma and cisplatin monotherapy were established by resazurin assay and used to design combination regimens. Treatment interactions were quantified by coefficient of drug interaction (CDI) analysis and monitored kinetically by long-term live-cell imaging. Plasma-derived ROS were measured in PBS and DMEM, and their functional relevance was assessed in SCaBER cells using catalase and N-acetylcysteine. In ovo validation was performed in the tumor chorioallantoic membrane (TUM-CAM) model, where tumor mass, vascularization, cellular marker expression, and cytokine secretion were analyzed. Results: Plasma and cisplatin exhibited opposing monotherapy sensitivity profiles across cell lines, creating a favorable basis for combination treatment. CDI analysis revealed clear synergy in SCaBER at intermediate cisplatin concentrations, additive effects in RT-112, and additive to mildly synergistic effects in T24. ROS profiling and scavenger experiments identified hydrogen peroxide as a key mediator of plasma and plasma–cisplatin cytotoxicity in SCaBER. In the TUM-CAM model, plasma and cisplatin monotherapies showed notable antitumoral potential. At the same time, plasma–cisplatin combination therapy elicited only modest effects on tumor growth and vascularization compared to monotreatments but induced distinct, cell line-specific alterations in cytokine and marker expression. Conclusions: These findings demonstrate that plasma can potentiate cisplatin cytotoxicity in bladder cancer cells and reshape tumor-associated molecular signatures, supporting further optimization and preclinical evaluation of plasma–cisplatin combination therapy. Full article

Background/Objectives: Thymol (THY) is widely used in oral care products for its antimicrobial and anti-inflammatory activity, but data on its cytocompatibility, potential differential effects on oropharyngeal-derived cells, and mucosal irritation under prolonged exposure remain limited. This study evaluated THY’s effects on healthy human gingival fibroblasts (HGF-1) and pharyngeal carcinoma (Detroit-562) cells after 24 h exposure, together with its irritation potential in ovo. Methods: Cells were treated with THY (100–300 µM) for 24 h. Cellular viability (MTT), morphology, mitochondrial alterations (MitoTracker™/Hoechst 33342), mitochondrial membrane potential (JC-1), and apoptosis/necrosis (AO/PI) were assessed. Clonogenic assays evaluated long-term proliferative capacity. Lastly, irritation score was examined using the HET-CAM assay at 300 µM. Results: THY produced a dose-dependent viability decrease in both lines, with HGF-1 viability remaining ≥75% and Detroit-562 reduced to ~68% at 300 µM. Morphology, mitochondrial staining, JC-1 ratios, and AO/PI imaging showed progressive apoptotic features, more evident in Detroit-562 cells. Clonogenic capacity increased slightly in HGF-1 at 100 µM and declined to ~75% at 300 µM, whereas Detroit-562 colonies decreased from ~68% to ~40% across the dose range. Additionally, THY (300 µM) showed no irritation in the HET-CAM assay. Conclusions: THY demonstrated acceptable cytocompatibility in gingival fibroblasts, stronger inhibitory effects on carcinoma cells at higher concentrations, and no acute irritation in ovo. These findings support THY’s safe use within defined concentration limits and justify further evaluation in advanced oral tissue models. Full article

The prevalence of diet-related noncommunicable diseases (NCDs; e.g., obesity, type 2 diabetes, cardiovascular disease, and certain cancers) is increasing globally, while food systems are also driving climate change and biodiversity loss. Transitioning to predominantly plant-based (“plant-forward”) dietary patterns can improve health and lower environmental impacts. We present the Slovenian Nutrition Guidelines 2025 (SNG2025)—their methodology, development, and core recommendations. Developed as adult food-based dietary guidelines, the SNG2025 are evidence-informed, drawing on the scientific literature, national nutritional data, and expert consensus. We set quantitative daily intake targets by integrating evidence on primary NCD outcomes with environmental metrics (greenhouse gas emissions, land use, and water use), which led to upper limits for animal-based foods. The recommended plant-forward dietary pattern, aligned with the EAT–Lancet planetary health diet, emphasises vegetables, fruits, legumes, whole grains, nuts, seeds, and unsaturated oils; allows low-to-moderate amounts of seafood, poultry, dairy, and eggs; and keeps red and processed meat, free sugars, refined grains, saturated fat, salt, ultra-processed foods (UPFs), and alcohol to a minimum. For the first time, we operationalise health, environmental sustainability, and equity (cultural diversity and accessibility) through a three-tier, plant-forward food plate model (Mediterranean, vegetarian [lacto-ovo], and whole food, plant-based [vegan]). The SNG2025 aim to reduce the risk of NCDs, lower the dietary environmental footprint, and improve fair access to healthy food. They signal a shift from disease management to a prevention-oriented, systems approach that aligns health and ecological goals. With robust implementation, supportive policies, and multisector collaboration, the SNG2025 can strengthen population health, foster more resilient food systems, and advance equity and long-term sustainability. Full article